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Interactions Between the Ur Element RNA Oligonucleotide and Cstf

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Interactions Between the Ur Element RNA Oligonucleotide and Cstf 1 Interactions Between the Ur Element RNA Oligonucleotide and CstF-64 in C. elegans Nematodes Bryan Nycz University of Colorado at Boulder Molecular, Cellular, and Developmental Biology Senior Honors Thesis April 5, 2013 Thesis Advisor: Dr. Thomas Blumenthal, MCDB Direct supervisor: Dr. Erika Lasda Committee Members: Dr. Jennifer Martin, MCDB Dr. James Goodrich, CHEM Dr. Nancy Guild, MCDB 2 Abstract In C. elegans, RNA processing of downstream operon genes through SL2 trans-splicing is crucial for polycistronic transcripts to be separated into monocistronic transcripts. The Ur element RNA sequence, which is important for proper SL2 trans-splicing, is found in the intercistronic region (ICR) of the pre-mRNA. The ICR is located between the 3’ end of one gene and the trans-splice site of a downstream operon gene. Lasda has shown that the Ur element sequence is required for downstream SL2 trans-splicing and proposed that it defines the 5’splice site on the SL2 RNA (Lasda, Allen, & Blumenthal, 2010). This project investigates whether there is an interaction between the Ur element RNA oligonucleotide and purified GST CstF-64, a 3’end formation factor. The Ur element RNA oligo contains short stem-loop and multiple UAYYUU motifs similar to Ur element RNA sequence found in ICRs. These experiments specifically analyze whether various mutant and wild type Ur element RNA oligos pull down GST CstF-64 directly, in the absence of extract. Results suggest that the wild type Ur element RNA oligo interacts directly with GST CstF-64, while the mutant RNA oligo does not pull down GST CstF-64. The results also suggest that the region containing the UAYYUU is required for GST CstF-64 binding, while the stem-loop is not important. Additionally, preliminary cross- linking results suggest that GST CstF-64 directly binds the loop and first UAYYUU motif of the Ur element RNA oligo. 3 Introduction In 1965, Sydney Brenner chose Caenorhabditis elegans, a nematode, for genetic analysis to study animal development and behavior (Hillier et al., 2005; Riddle, Blumenthal, Meyer, & Priess, 1997). He settled on this organism because of its small size, rapid life cycle, and minimal laboratory cultivation complexity. Specifically, a single nematode can produce between 300 to 350 offspring. Furthermore and most importantly, C. elegans was selected because of its anatomical simplicity, less than 1,000 cells, and small genome (Riddle et al., 1997). Recent sequence analysis has uncovered that C. elegans’ 100,291,840 base pair (bp) genome contains 19,735 protein-coding genes and roughly 1,300 non-coding RNA genes (Hillier et al., 2005). Because of this simplicity and the sheer amount of information present on this species, C. elegans has become a preferred model organism in molecular and cellular biological studies (Riddle et al., 1997). While studying this species, researchers discovered that C. elegans utilize an uncommon mechanism, similar to what is observed in trypanosomes and other lower metazoans, to process their pre-mRNA into a mature transcript (Riddle et al., 1997; Spieth, Brooke, Kuersten, Lea, & Blumenthal, 1993). This unique mechanism is known as SL trans-splicing, and involves joining together two separate RNA molecules. In C. elegans, 70% of pre-mRNA is SL trans-spliced (Blumenthal, 2012). Specifically, SL trans-splicing involves joining the spliced leader (SL), which is a 22 nucleotide (nt) conserved leader sequence donated by a longer SL RNA, to the 5’ end of the first exon in the pre-mRNA (Lasda & Blumenthal, 2011). In C. elegans, there are two different spliced leader sequences, SL1 and SL2. SL1 RNAs, donate their mini-exon, the SL, to the first exon in polycistrons, as well as non-operon genes, whereas SL2 RNAs, donate their mini-exon to downstream genes in operons (Lasda et al., 2010). Operons in C. elegans are 4 polycistronic transcripts containing clusters of up to eight genes transcribed from a single promoter (Blumenthal, 2012; Lasda et al., 2010). In operons, SL trans-splicing is responsible for transcript separation into monocistronic transcripts (Riddle et al., 1997). The SL RNA molecules are similar to U small nuclear RNAs (snRNAs). They bear a hypermethylated m2,2,7GpppN trimethylguanosine (TMG) 5’ cap, and are predicted to form stem-loop secondary structures through intramolecular base pairing. Additionally, SL RNAs contain Sm binding sites, where Sm proteins important in splicing bind (Lasda & Blumenthal, 2011; Thomas, Conrad, & Blumenthal, 1988). Addition of the spliced leader serves two important purposes: it provides the cap to mRNAs in downstream genes and functions jointly with polyadenylation in separating polycistronic transcripts (Thomas et al., 1988). In C. elegans, the mechanism for trans-splicing is considered to be similar to that of cis- splicing. Cis-splicing is a process that involves intron removal and joining adjacent exons together. In the first step, the upstream exon is cut from the intron and a lariat-like intermediate molecule is produced. The two exons are then joined together by a phosphodiester strand-transfer reaction removing the intron. Important to this reaction are five snRNAs and numerous proteins. These small nuclear ribonucleoprotien (snRNP) complexes and associated proteins make up the spliceosome. The snRNPs include U1, U2, U4, U5, and U6, and are responsible for the splicing reaction (Blumenthal & Steward, 1997). Analogous to cis-splicing, trans-splicing progresses through a two-step trans- esterification reaction ( Liang, Haritan, Uliel, & Michaeli, 2003). Furthermore, trans-splicing uses the same splice site consensus sequences, is catalyzed by most of the identical snRNPs, and involves a branched intermediate (Spieth et al., 1993). Because of these similarities, it has been proposed that trans-splicing evolved from cis-splicing (Blumenthal, 1995, 2004; Lasda & 5 Blumenthal, 2011). Conversely, three notable differences between these two mechanisms are that trans-splicing does not involve U1 snRNP and it produces a Y-branched product, whereas cis- splicing produces a lariat product (Lasda et al., 2010; Spieth et al., 1993). Additionally, SL RNAs in trans-splicing are “consumed” and not recycled like the other spliceosomal snRNPs. This happens because the mini-exon is separated from the SL RNA and remains connected to the mRNA (Lasda & Blumenthal, 2011). All genes in operons and non-operons are processed by RNA cleavage and polyadenylation at the 3’ end. Specifically, this is known as RNA 3’ end formation. This processing contributes to mature mRNA stability and transport. Although roughly eighty-five proteins are involved in 3’ end formation, the core machinery is comprised of the cleavage- polyadenylation specificity factor complex (CPSF), poly(A) polymerase (PAP), and the cleavage stimulation factor complex (CstF) (Shi et al., 2009; Takagaki & Manley, 1997). Both the CPSF complex and the CstF complex are required for proper cleavage, while PAP is required for the addition of the poly(A) tail. Specifically, CstF is a heterotrimeric complex composed of three protein subunits: 77, 64, and 50. CstF-77 bridges CstF-64 and CstF-50 and also interacts with CPSF 160, one of the four subunits of CPSF. CstF-64 directly binds RNA sequences, and CstF-50 directly contacts the carboxy terminal domain (CTD) on RNA polymerase II (RNAPII) (Colgan & Manley, 1997). It has also been observed that CstF-77, as well as CstF-50, can self-associate. Because of this association, Takagaki and Manley (1997) have also suggested that multiple CstF complexes might dimerize during 3’ end formation. The two primary pre-mRNA sequences required for cleavage and polyadenylation are a AAUAAA sequence upstream of the cleavage site that CPSF 160 of the CPSF complex binds, 6 and a GU or U rich sequence downstream of the cleavage site that CstF-64 of the CstF complex binds (Takagaki & Manley, 1997). CstF-64 also interacts with symplekin, a subunit of CPSF, and Pcf11, a subunit of Cleavage Factor (CF) IIm (Chan, Choi, & Shi, 2011). In C. elegans internal operon genes, there are two U-rich regions downstream of cleavage sites and it is not known to which, if either or both, CstF-64 binds (Graber, Salisbury, Hutchins, & Blumenthal, 2007). This is important because this second U rich region contains the Ur element sequence required for SL2 trans-splicing, discussed below. A key element involved in SL2 trans-splicing is the Ur (U-rich) element found within the intercistronic region (ICR) between any two genes of the same operon. The Ur element sequence is around 50 nt upstream of the SL2 trans-splice site in the pre-mRNA (Lasda et al., 2010). The Ur element is composed of a short stem-loop followed by a UAYYUU (where Y represents a Cytosine or Uracil) sequence (Lasda et al., 2010). The UAYYUU sequence is frequently followed by multiple overlapping UAYYUU motifs. The presence of the stem is conserved among operons, but the primary sequence of the stem may not be. This element is required for downstream SL2 trans-splicing. Huang et al. (2001) showed that when the Ur element was mutated, the amount of downstream mRNA diminished. As well, it was observed that without the Ur element, there was a total disappearance of SL2 trans-splicing to gpd mRNA (Huang et al., 2001). Lasda et al. (2010) showed that in-vitro, mutation of the Ur element led to a loss of SL2 trans-splicing, without a concomitant increase in SL1 trans-splicing. Huang et al. (2001) suggested that the Ur element might contain or act as a protein binding site and contribute to SL2 trans-splicing. By creating RNA oligos that were derived from the Ur element sequence in the ICR preceding the gene rla-1, Lasda et al. (2010) were able to test the correlation between SL2 7 activation and the Ur element sequence. These oligos are 49 nucleotides and contain the stem- loop region followed by all of the overlapping copies of the UAYYUU motif (see Figure 1A).
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